U.S. patent application number 10/677980 was filed with the patent office on 2005-10-27 for plasmodium falciparum erythrocyte binding protein baebl for use as a vaccine.
Invention is credited to Mayer, Ghislaine, Miller, Louis H..
Application Number | 20050239730 10/677980 |
Document ID | / |
Family ID | 23076065 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050239730 |
Kind Code |
A1 |
Mayer, Ghislaine ; et
al. |
October 27, 2005 |
Plasmodium falciparum erythrocyte binding protein baebl for use as
a vaccine
Abstract
The invention relates to Plasmodium falciparum Erythrocyte
Binding Protein BAEBL for use as a vaccine.
Inventors: |
Mayer, Ghislaine;
(Gaithersburg, MD) ; Miller, Louis H.; (Rockville,
MD) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
23076065 |
Appl. No.: |
10/677980 |
Filed: |
October 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10677980 |
Oct 2, 2003 |
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PCT/US02/10071 |
Mar 29, 2002 |
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60281130 |
Apr 2, 2001 |
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Current U.S.
Class: |
514/44R ;
424/191.1 |
Current CPC
Class: |
A61P 33/06 20180101;
Y02A 50/412 20180101; C07K 14/445 20130101; A61K 39/00
20130101 |
Class at
Publication: |
514/044 ;
424/191.1 |
International
Class: |
A61K 048/00; A61K
039/002 |
Claims
What is claimed is:
1. A vaccine composition comprising a polypeptide and a
pharmaceutically acceptable vehicle, wherein the polypeptide
comprises an amino acid sequence that encodes a BAEBL polypeptide
or portion thereof.
2. A vaccine composition of claim 1, wherein the polypeptide
portion is an amino acid sequence that encodes a BAEBL region II or
portion thereof.
3. A vaccine composition of claim 2, wherein the polypeptide
portion is selected from the group consisting of an amino acid
sequence having the following number of consecutive amino acids
taken from said BAEBL polypeptide: 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,
152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,
165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,
178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,
243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,
256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,
269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,
282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,
295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,
308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,
321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,
334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,
347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,
360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,
373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,
386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,
399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,
412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,
425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,
438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,
451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463,
464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,
477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,
490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502,
503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515,
516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528,
529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541,
542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554,
555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567,
568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580,
581, 582, 583, and 584.
4. A vaccine composition of any of claims 1-3 wherein said BAEBL
polypeptide or portion thereof is defined as having the amino acid
sequence of SEQ ID NO: 2 or portion thereof.
5. A vaccine composition of any of claims 1-3 wherein said BAEBL
polypeptide or portion thereof is defined as having at least 70%,
80%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID
NO: 2 or portion thereof.
6. A vaccine composition of any of claims 1-3 wherein said BAEBL
polypeptide or portion thereof is encoded by a polynucleotide
defined as having at least 70%, 80%, 90%, 95%, or 99% identity to
the open reading frame of SEQ ID NO: 1 or portion thereof.
7. A vaccine composition of any of claims 1-3 wherein said BAEBL
polypeptide or portion thereof is encoded by a polynucleotide which
hybridizes at 42 degree C. in a solution comprising: 50% formamide,
5 times SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 5 times Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA,
followed by washing the filters in 0.1 times SSC at about 65 degree
C., to a second polynucleotide having the polynucleotide sequence
of SEQ ID NO: 1.
8. A vaccine composition of any of claims 1-3, wherein said BAEBL
polypeptide or portion thereof has a polymorphism selected from the
group consisting of I at position 185, N at position 239, T at
position 261, R at position 261, and E at position 285.
9. A vaccine composition of any of claims 1-3 further comprising an
adjuvant selected from the group consisting of QS-21, Detox-PC,
MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B,
Adjumer, PG-026, GSK-1, GcMAF, B-alethine, MPC-026, Adjuvax, CpG
ODN, Betafectin, Alum, and MF59.
10. A vaccine composition of any of claims 1-3 further comprising a
second polypeptide, wherein said second polypeptide comprises an
amino acid sequence that encodes at least a portion of a Duffy
binding protein or erythrocyte binding antigen-175 (EBA-175) of a
malaria Plasmodium parasite.
11. A vaccine composition comprising a polynucleotide and a
pharmaceutically acceptable vehicle, wherein the polynucleotide
comprises a nucleic acid sequence that encodes a BAEBL polypeptide
or portion thereof.
12. A vaccine composition of claim 11, wherein the polypeptide
portion is an amino acid sequence that encodes a BAEBL region II or
portion thereof.
13. A vaccine composition of claim 12, wherein the polypeptide
portion is selected from the group consisting of an amino acid
sequence having the following number of consecutive amino acids
taken from said BAEBL polypeptide: 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,
152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,
165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,
178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,
243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,
256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,
269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,
282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,
295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,
308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,
321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,
334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,
347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,
360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,
373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,
386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,
399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,
412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,
425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,
438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,
451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463,
464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,
477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,
490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502,
503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515,
516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528,
529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541,
542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554,
555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567,
568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580,
581, 582, 583, and 584.
14. A vaccine composition of any of claims 11-13 wherein said BAEBL
polypeptide or portion thereof is defined as having the amino acid
sequence of SEQ ID NO: 2 or portion thereof.
15. A vaccine composition of any of claims 11-13 wherein said BAEBL
polypeptide or portion thereof is defined as having at least 70%,
80%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID
NO: 2 or portion thereof.
16. A vaccine composition of any of claims 11-13 wherein said BAEBL
polypeptide or portion thereof is encoded by a polynucleotide which
is identical to the open reading frame of SEQ ID NO: 1 or portion
thereof.
17. A vaccine composition of any of claims 11-13 wherein said BAEBL
polypeptide or portion thereof is encoded by a polynucleotide
defined as having at least 70%, 80%, 90%, 95%, or 99% identity to
the open reading frame of SEQ ID NO: 1 or portion thereof.
18. A vaccine composition of any of claims 11-13 wherein said BAEBL
polypeptide or portion thereof is encoded by a polynucleotide which
hybridizes at 42 degree C. in a solution comprising: 50% formamide,
5 times SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 5 times Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA,
followed by washing the filters in 0.1 times SSC at about 65 degree
C., to a second polynucleotide having the polynucleotide sequence
of SEQ ID NO: 1.
19. A vaccine composition of any of claims 11-13, wherein said
BAEBL polypeptide or portion thereof has a polymorphism selected
from the group consisting of I at position 185, N at position 239,
T at position 261, R at position 261, and E at position 285.
20. A method of vaccinating a human against a malaria Plasmodium
parasite comprising the step of administering the vaccine
composition of any of claims 1-3 or 11-13 to said human.
21. The method of claim 20 wherein said step of administration is
by protein immunization.
22. The method of claim 20 wherein said step of administration is
by genetic immunization.
23. A method of vaccinating a human against a malaria Plasmodium
parasite comprising the step of administering antibodies specific
for the binding site of a BAEBL ligand in an amount sufficient to
inhibit the ligand from binding red blood cells in the human.
Description
RELATED APPLICATIONS
[0001] This application is a continuation and claims the benefit of
priority of International Application No. PCT/US02/10071 filed Mar.
29, 2002, designating the United States of America and published in
English, which claims the benefit of priority of U.S. Provisional
Application No. 60/281,130 filed Apr. 2, 2001, both of which are
hereby expressly incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The invention relates to Plasmodium falciparum Erythrocyte
Binding Protein BAEBL for use as a vaccine.
BACKGROUND OF THE INVENTION
[0003] The erythrocytic stage of Plasmodium falciparum causes
several million deaths yearly, primarily in Africa. The parasite
lives within the erythrocyte except during the brief period when
merozoites, the invasive stage of the parasite, are released from
infected erythrocytes to invade uninfected erythrocytes. Invasion
of erythrocytes by merozoites is a multistep process that includes:
attachment, reorientation of the merozoite in such a way that its
apical end is in contact with the erythrocyte surface, junction
formation, and entry into the parasitophorous vacuole (Dvorak, J.
A. et al. 1975 Science 187: 748-9; Aikawa, M. et al. 1978 J Cell
Biol 77: 72-82). The binding of merozoites to erythrocytes requires
parasite receptors (Camus, D. & Hadley, T. H. 1985 Science 230:
553-556; Haynes, J. D. et al. 1988 J Exp Med 167: 1873-1881; Adams,
J. H. et al. 1990 Cell 63: 142-153; Sim, B. K. L. et al. 1990 J
Cell Biol 111: 1877-1884; Galinski, M. R. et al. 1992 Cell 69:
1213-1226).
[0004] One family of these parasite receptors is named Duffy
binding-like erythrocyte binding protein (DBL-EBP) for its
similarity to the P. vivax and P. knowlesi proteins that bind to
the Duffy blood group antigens (Duffy positive) on human
erythrocytes (Adams, J. H. et al. 1992 PNAS USA 89: 7085-7089). P.
vivax does not infect Africans lacking the Duffy blood group
antigens (Duffy negative), and P. knowlesi will not form a junction
with or invade Duffy negative human erythrocytes (Miller, L. H. et
al. 1976 N Engl J Med 295: 302-304; Miller, L. H., et al. 1979 J
Exp Med 149: 172-184). Region II, a domain of the P. vivax DBL-EBP,
has the same specificity as the full-length protein (Chitnis, C. E.
& Miller, L. H. 1994 J Exp Med 180: 497-506).
[0005] P. knowlesi has three highly homologous DBL proteins, each
with different specificities as defined by region II (Haynes, J. D.
et al. 1988 J Exp Med 167: 1873-1881; Ranjan, A. & Chitnis, C.
E. 1999 PNAS USA 96: 14067-14072). One binds to Duffy blood group
antigens on human and rhesus erythrocytes, a second binds to sialic
acid on rhesus erythrocytes, and a third binds to an unidentified
receptor on rhesus erythrocytes. Whereas P. knowlesi can only
invade Duffy positive human erythrocytes, it can invade rhesus
erythrocytes that have been rendered Duffy negative by protease
treatment and by removal of sialic acid with neuraminidase (Haynes,
J. D. et al. 1988 J Exp Med 167: 1873-1881; Miller, L. H. et al.
1973 J Exp Med 138: 1597-1601). P. knowlesi invades these
enzymatically treated erythrocytes at the same rate as the
untreated erythrocytes, indicating a highly efficient alternative
pathway of invasion.
[0006] The Duffy binding proteins of P. vivax (PvDBP) and P.
knowlesi (PkDBP) are part of a larger family of Plasmodium proteins
that include EBA-175 of P. falciparum. EBA-175 binds to sialic acid
and the peptide backbone of glycophorin A on the erythrocyte
surface (Sim, B. K. L. et al. 1994 Science 264: 1941-1944). As in
the case of P. vivax, the binding domain of EBA-175 is defined by
region II. Unlike P. vivax, which cannot infect Duffy negative
erythrocytes, some strains of P. falciparum parasites have
alternative pathways of invasion, not requiring glycophorin A for
either invasion or growth in vitro. Thus, other receptors must be
involved in these alternative pathways (Dolan, S. A. et al. 1990 J
Clin Invest 86: 618-624).
[0007] The P. falciparum genome sequence identifies at least four
paralogues of EBA-175. We have studied one of these DBL genes of P.
falciparum, named baebl (Adams, J. H. et al. 2001 Trends Parasitol
17: 297-9), to explore its possible role in invasion.
SUMMARY OF THE INVENTION
[0008] The present invention relates to Plasmodium falciparum
Erythrocyte Binding Protein BAEBL for use as a vaccine. A BAEBL
polynucleotide sequence or a portion thereof, or a BAEBL
polypeptide sequence or a portion thereof, is used to induce an
immune response to a Plasmodium parasite, whereby a human is
protected against malaria. The BAEBL polynucleotide sequence is
alternatively used to express recombinant polypeptides or portions
thereof. Furthermore, synthetic BAEBL polypeptides or portions
thereof are prepared in accordance with the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0010] FIG. 1. Sequencing strategy and exon/intron structure of
baebl. Oligonucleotides were designed based on the genomic sequence
obtained from the Plasmodium falciparum genome project (Sanger
Centre) and used for sequencing of genomic DNA (GenBank No.
AF332918) and RT-PCR of mRNA (GenBank No. AF332919) to determine
the intron/exon structure in P. falciparum Dd2/Nm strain. (A)
Schematic representation of the gene and predicted protein
structure of baebl. Predicted protein structure has strong
similarity with EBA-175, containing the putative signal sequence
(SS; aa 1-21) predicted by SIGNALP V2.0; region II (two Duffy
binding-like (DBL) domains, F1 and F2); region VI (3.degree. Cys),
the transmembrane domain (TM; aa 1134-1153) predicted by TMHMM
V2.0, followed by the putative cytoplasmic domain (Cyt). (B) f1 to
f9 primers (see Examples) are used for RT-PCR of mRNA (lanes marked
c) and PCR of genomic DNA (lanes marked g). Bar=1 kb.
[0011] FIG. 2. Confocal microscopy demonstrates the localization of
BAEBL in micronemes. (A) Dd2/Nm schizonts were double labeled with
anti-BAEBL region II and anti-EBA-175. Schizonts immunolabeled with
anti-BAEBL region II were stained with Alexa 488 secondary
antibody. Schizonts labeled with anti-EBA-175 were stained with
Alexa 594 secondary antibody. (B) Dd2/Nm schizonts were double
labeled with anti-BAEBL region VI and anti-EBA-175. Schizonts
immunolabeled anti-BAEBL region VI were stained with Alexa 488
secondary antibody. Schizonts labeled with anti-EBA-175 were
stained with Alexa 594 secondary antibody. (C) Dd2/Nm schizonts
were double labeled with anti-BAEBL region II and anti-RAP-1
monoclonal antibody. Schizonts immunolabeled anti-BAEBL region II
were stained with Alexa 488 secondary antibody. Schizonts labeled
with anti-RAP-1 were stained with TRITC secondary antibody. (D)
Dd2/Nm schizonts were double labeled with anti-BAEBL region VI and
anti-RAP-1 monoclonal antibody. Schizonts immunolabeled with
anti-BAEBL region VI were stained with Alexa 488 secondary
antibody. Schizonts labeled with anti-RAP-1 were stained with TRITC
secondary antibody.
[0012] FIG. 3. Evidence that anti-region II (Anti-R2) and
anti-region VI (Anti-R6) sera immunoprecipitate the same protein.
The supernatant was preabsorpted with either anti-region 2 or
anti-region 6 followed by immunoprecipitation by the two sera.
BAEBL was removed by both sera.
[0013] FIG. 4. BAEBL and EBA-175 did not bind to neuraminidase (NM
eluate) or trypsin-treated erythrocytes (Trypsin RBC). Eluates of
BAEBL and EBA-175 were only seen from normal erythrocytes.
[0014] FIG. 5. BAEBL binds and is eluted from En(a-) erythrocytes
that lack glycophorin A. NM RBC are neuraminidase-treated normal
erythrocytes.
[0015] FIG. 6. Absorption and elution of BAEBL (A, B) and EBA-175
(C, D) with various amounts (25, 50, 2.times.50 .mu.l, and
4.times.50 .mu.l of packed erythrocytes) of Gerbich [Ge(-2, -3,
4)], normal, and neuraminidase (NM)-treated erythrocytes. For
elution, 25 .mu.l and 50 .mu.l of packed erythrocytes were
used.
SUMMARY OF SEQUENCES
[0016] SEQ ID NO: 1 and FIG. 7 are the genomic sequence encoding
Plasmodium falciparum Erythrocyte Binding Protein BAEBL; start and
stop codons are indicated in bold, and the introns span nucleotides
3499-3638, 3718-3846, and 3930-4061.
[0017] SEQ ID NO: 2 is the amino acid sequence encoding Plasmodium
falciparum Erythrocyte Binding Protein BAEBL.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0018] A member of a Plasmodium receptor family for erythrocyte
invasion was identified on chromosome 13 from the Plasmodium
falciparum genome sequence of the Sanger Centre. The protein (named
BAEBL) has homology to EBA-175, a P. falciparum receptor that binds
specifically to sialic acid and the peptide backbone of glycophorin
A on erythrocytes. Both EBA-175 and BAEBL localize to the
micronemes, an organelle at the invasive end of the parasite that
contains other members of the family. Like EBA-175, the erythrocyte
receptor for BAEBL is destroyed by neuraminidase and trypsin,
indicating that the erythrocyte receptor is a sialoglycoprotein.
Its specificity, however, differs from EBA-175 in that BAEBL can
bind to erythrocytes that lack glycophorin A, the receptor for
EBA-175. It has reduced binding to erythrocytes with the Gerbich
mutation found in another erythrocyte sialoglycoprotein
(glycophorin C/D). The interest in BAEBL's reduced binding to
Gerbich erythrocytes derives from the high frequency of the Gerbich
phenotype in some regions of Papua New Guinea where P. falciparum
is hyperendemic.
[0019] The present invention relates, in general, to a
substantially purified polynucleotide sequence (e.g. a DNA
sequence) encoding all, or a portion, of BAEBL of the DBL-EBP
family of a Plasmodium parasite (particularly, Plasmodium
falciparum). A "portion" as used herein preferably consists of at
least five (or six) amino acids or, correspondingly, at least 15
(or 18) nucleotides. GenBank No. AF332918 encodes the genomic DNA
and GenBank No. AF332919 encodes the cDNA of BAEBL.
[0020] The present invention further relates to a polynucleotide
sequence encoding a BAEBL protein of other Plasmodium parasites
such as, for example, P. vivax or P. knowlesi. One of ordinary
skill in the art, given the present disclosure, could easily
identify and clone analogous genes in such species without undue
experimentation.
[0021] In one embodiment, the present invention relates to a
polynucleotide sequence given in SEQ ID NO: 1 encoding the entire
amino acid sequence of BAEBL (the specific DNA sequence defined
therein being only an example). The polynucleotide sequence can be
genomic DNA or cDNA. Polynucleotide sequences to which this
invention relates also include those encoding substantially the
same protein as that encoded by SEQ ID NO: 1, which include, for
example, allelic forms of the given amino acid sequences and
alternatively spliced products.
[0022] The present invention relates to a recombinant DNA molecule
comprising a vector and a DNA sequence encoding BAEBL, or a portion
thereof. Using methodology well known in the art, recombinant DNA
molecules of the present invention can be constructed. Possible
vectors for use in the present invention include expression
vectors. The Plasmodium BAEBL encoding sequences of the present
invention can be inserted into commercially available DNA vectors
(expression vectors) to express the encoded protein product. The
expression vectors have promoter sequences and other regulatory
sequences necessary for expression in host cells. The technique of
using expression vectors to introduce exogenous genes and express
their protein products in a host cell is well known to those
familiar with the art. For example the expression vector pET21a is
commercially available and can be used to express proteins in E.
coli. Alternatively the protein can be expressed in a eukaryotic
cell, such as yeast, using Pichia expression vectors (i.e. pHIL-D2)
commercially available from Invitrogen. The baculovirus system is
also commercially available and can be used to express the BAEBL
genes in insect cultures.
[0023] Once the baebl gene or fragment thereof has been cloned into
an expression vector, the resulting vector can be used to transform
a host cell, using procedures known to those familiar with the art.
Such transformation procedures include but are not limited to
microinjection, microprojectile bombardment, electroporation,
calcium chloride permeabilization, polyethylene glycol
permeabilization, protoplast fusion or bacterial mediated
mechanisms such as Agrobacterium tumafaciens or Agrobacterium
rhizogenes.
[0024] Host cells may be selected from any cell in which expression
of modified proteins can be made compatible, including bacteria,
fungus, yeast, plant cells and animal cells. Suitable host cells
include prokaryotes selected from the genus Escherichia or
Staphylococcus and eukaryotes selected from the genus Pichia
(including Saccharomyces cerevisae, for example). In addition,
mammalian cell culture (such as CHO and COS cells) can be used to
express the BAEBL proteins and peptide fragments.
[0025] The transformed host cells synthesize the BAEBL protein or
peptide fragment which can be isolated and purified using standard
methods known to those familiar with the art. In one embodiment the
BAEBL proteins and peptide fragments can be expressed as fusion
proteins to assist in the purification of the BAEBL protein
products.
[0026] The present invention also relates to a Plasmodium BAEBL
protein separated from those proteins with which it is naturally
associated. One skilled in the art can easily purify BAEBL using
methodologies well known in the art.
[0027] The present invention further relates to a recombinantly
produced BAEBL protein with the amino acid sequence given in SEQ ID
NO: 2, an allelic variation thereof or a chimeric protein thereof.
The present invention also relates to recombinantly produced
peptide fragments of BAEBL. Further, the present invention relates
to synthetic BAEBL or a synthetic peptide fragment thereof.
[0028] The present invention further relates to a polypeptide
comprising an amino acid sequence having a consecutive number of
amino acid sequences selected from a BAEBL protein, for example,
Plasmodium BAEBL protein having the sequence of SEQ ID NO: 2, which
are useful as diagnostic agents or can be utilized as therapeutic
agents for treating or preventing malaria. Some agents are useful
as antigenic fragments to display linear epitopes, and other agents
are useful as antigenic fragments to display conformational
epitopes for generating neutralizing antibodies. In another
embodiment, the invention relates to a polypeptide comprising an
amino acid sequence that encodes an EBA-175-like domain of a BAEBL
protein, which is BAEBL region II, constituting two Duffy
binding-like (DBL) domains, F1 and F2, for example, Plasmodium
BAEBL protein having the sequence of SEQ ID NO: 2 running from
amino acids 154-738. In another embodiment, the invention is
directed to a polypeptide comprising an amino acid sequence having
the following number of consecutive amino acids taken from a BAEBL
protein, for example, Plasmodium BAEBL protein having the sequence
of SEQ ID NO: 2: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,
129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,
142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,
168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,
181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,
194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,
207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,
220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,
246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,
259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,
272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,
285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,
298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,
311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,
324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,
337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,
350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,
363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375,
376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,
389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401,
402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,
415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427,
428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,
441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,
454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,
467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,
480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,
493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505,
506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518,
519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531,
532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544,
545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557,
558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570,
571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583,
and 584.
[0029] The present invention further relates to antibodies specific
for epitopes present on the BAEBL proteins of the Plasmodium
parasites. Thus, in one embodiment, the present invention relates
to antibodies (such as monoclonal, polyclonal, chimeric, humanized,
and anti-idiotypic) specific for the BAEBL protein. More
particularly, the antibodies are directed to conserved regions of
BAEBL and more preferably to BAEBL region II. One skilled in the
art, using standard techniques well known to those skilled in the
art can raise antibodies to the proteins and peptide fragments
disclosed in the present invention. These antibodies can be useful
as diagnostic agents or can be utilized as therapeutic agents for
treating or preventing malaria.
[0030] The present invention relates to a vaccine for use in humans
against malaria. As is customary for vaccines, BAEBL or a portion
thereof, can be delivered to a human in a pharmacologically
acceptable vehicle. As one skilled in the art will understand, it
is not necessary to use the entire protein. A portion of the
protein (for example, a peptide corresponding to a conserved region
of the BAEBL protein) can be conjugated to pharmacologically
acceptable carriers, including diphtheria toxoid, pertussis toxoid,
or tetanus toxoid.
[0031] Vaccines of the present invention can include effective
amounts of immunological adjuvants known to enhance an immune
response. Adjuvants suitable for co-administration in accordance
with the present invention should be ones that are potentially
safe, well tolerated and effective in people. Such immunological
adjuvants include QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G,
CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-1, GcMAF,
B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59
(see Kim et al. 2000 Vaccine, 18: 597 and references therein).
[0032] The protein or peptide fragment is present in the vaccine in
an amount sufficient to induce an immune response against the
antigenic portion and thus to protect against Plasmodium infection
thereby protecting the human against malaria. Protective antibodies
are usually best elicited by a series of 2-3 doses given about 2 to
3 weeks apart. The series can be repeated when concentrations of
circulating antibodies in the human drops. Further, the vaccine can
be used to immunize a human against other forms of malaria (that
is, heterologous immunization).
[0033] Compositions comprising substantially purified
polynucleotide sequences encoding a BAEBL protein or peptide
fragment can be used in accordance with the present invention as
vaccines. Live vector viruses are contemplated, where retroviruses,
adenoviruses, or adeno-associated viruses are engineered to carry
polynucleotide sequences encoding a BAEBL protein or peptide
fragment. Genetic immunization is an alternative, where naked DNA
encoding a BAEBL protein or peptide fragment is administered to
cells and the encoded protein antigens are expressed.
[0034] The present invention further yet relates to receptor
blocking therapy which disrupts the life cycle of the parasite in
humans. Administering to a human antibodies of the present
invention specific for the binding site of the BAEBL ligand of the
present invention can prevent invasion of red blood cells by the
merozoite, a necessary event in the life cycle of the Plasmodium
parasite. Alternatively, the erythrocyte receptor can be
administered to a human, which is glycophorin C/D (Reid and Spring,
1994 Transfusion Medicine 4: 139). The BAEBL ligand on the
merozoite will bind the circulating receptor rather than the
determinate on the red blood cells. Attachment of the merozoite to
the red blood cells, and hence invasion of the parasite, is
prevented.
[0035] The major human malaria parasite, P. falciparum, has
redundant or alternate receptor-ligand pathways of invasion.
Therefore, an effective vaccine for blocking parasite invasion of
erythrocytes by P. falciparum malaria will also target the
redundant receptor ligand interactions that occur during the
invasion process. Thus in some embodiments, the present vaccine
compositions comprise a BAEBL polypeptide in combination with
additional Plasmodium specific proteins or peptide fragments. For
example, the second polypeptide may comprise an amino acid sequence
that encodes a Duffy binding protein or erythrocyte binding
antigen-175 (EBA-175) of a malaria Plasmodium parasite. The Duffy
binding protein and EBA-175 are members of the EBL family of
proteins that are utilized by Plasmodium parasites to invade
erythrocytes. Thus, one vaccine composition in accordance with the
present invention comprises two or more proteins (or peptide
fragments) and a pharmaceutically acceptable vehicle, wherein at
least one protein (or peptide fragment) is Duffy binding protein or
EBA-175. EBA-175 and Duffy binding proteins of Plasmodium parasites
have been described in the prior art as well as their use in
preparing vaccines to prevent malaria infections. See U.S. Pat Nos.
5,198,347 and 6,120,770.
[0036] The present invention also relates to a method of
vaccinating a vertebrate species, particularly a human, against a
malaria Plasmodium parasite. The method comprises the steps of
administering a vaccine composition comprising a protein or peptide
fragment of BAEBL where the peptide fragment comprises at least a
consecutive six amino acid sequence and a physiologically
acceptable vehicle. In one embodiment the vaccine composition
further comprises a second protein or peptide fragment wherein the
second protein or peptide fragment comprises the Duffy binding
protein or erythrocyte binding antigen-175 of an erythrocyte
binding protein. The vaccine composition also can include various
adjuvants known to those skilled in the art. The vaccine
composition can be administered to a vertebrate species either
orally or parenterally using techniques well known to those skilled
in the art.
[0037] Nucleic Acid Molecules
[0038] As indicated herein, nucleic acid molecules of the present
invention may be in the form of RNA or in the form of DNA obtained
by cloning or produced synthetically. The DNA may be
double-stranded or single-stranded. Single-stranded DNA or RNA may
be the coding strand, also known as the sense strand, or it may be
the non-coding strand, also referred to as the anti-sense
strand.
[0039] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0040] Nucleic acid molecules of the present invention include DNA
molecules comprising an open reading frame (ORF) of a wild-type
baebl gene; and DNA molecules which comprise a sequence
substantially different from those described above but which, due
to the degeneracy of the genetic code, still encode an ORF of a
wild-type BAEBL polypeptide. Of course, the genetic code is well
known in the art. Degenerate variants optimized for human codon
usage are preferred.
[0041] In another aspect, the invention provides a nucleic acid
molecule comprising a polynucleotide which hybridizes under
stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above. By "stringent hybridization conditions" is
intended overnight incubation at 42.degree. C. in a solution
comprising: 50% formamide, 5 times SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 times
Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1 times SSC at about 65 degree C.
[0042] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 nt of the
reference polynucleotide.
[0043] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide. Of
course, a polynucleotide which hybridizes only to a complementary
stretch of T (or U) resides, would not be included in a
polynucleotide of the invention used to hybridize to a portion of a
nucleic acid of the invention, since such a polynucleotide would
hybridize to any nucleic acid molecule containing a poly T (or U)
stretch or the complement thereof (e.g., practically any
double-stranded DNA clone).
[0044] As indicated herein, nucleic acid molecules of the present
invention which encode a BAEBL polypeptide may include, but are not
limited to those encoding the amino acid sequence of the
full-length polypeptide, by itself, the coding sequence for the
full-length polypeptide and additional sequences, such as those
encoding a leader or secretory sequence, such as a pre-, or pro- or
prepro-protein sequence, the coding sequence of the full-length
polypeptide, with or without the aforementioned additional coding
sequences, together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing, including
splicing and polyadenylation signals, for example, ribosome binding
and stability of mRNA; and additional coding sequence which codes
for additional amino acids, such as those which provide additional
functionalities.
[0045] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the BAEBL protein. Variants may
occur naturally, such as a natural allelic variant. By an "allelic
variant" is intended one of several alternate forms of a gene
occupying a given locus on a genome of an organism (Genes II, 1985
Lewin, B., ed., John Wiley & Sons, New York). Non-naturally
occurring variants may be produced using art-known mutagenesis
techniques.
[0046] Such variants include those produced by nucleotide
substitutions, deletions or additions, which may involve one or
more nucleotides. The variants may be altered in coding regions,
non-coding regions, or both. Alterations in the coding regions may
produce conservative or non-conservative amino acid substitutions,
deletions or additions. Especially preferred among these are silent
substitutions, additions and deletions, which do not alter the
properties and activities of the BAEBL polypeptide or portions
thereof Also especially preferred in this regard are conservative
substitutions.
[0047] Further embodiments of the invention include nucleic acid
molecules comprising a polynucleotide having a nucleotide sequence
at least 70% identical, and more preferably at least 80%, 90%, 95%
or 99% identical to a nucleotide sequence encoding a polypeptide
having the amino acid sequence of a wild-type BAEBL polypeptide or
a nucleotide sequence complementary thereto.
[0048] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a BAEBL polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence encoding the BAEBL polypeptide. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mutations of the reference sequence
may occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0049] As a practical matter, whether any particular nucleic acid
molecule is at least 70%, 80%, 90%, 95% or 99% identical to the
reference nucleotide sequence can be determined conventionally
using known computer programs such as the Bestfit program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, 575 Science Drive,
Madison, Wis. 53711). Bestfit uses the local homology algorithm of
Smith and Waterman, Advances in Applied Mathematics 2: 482-489
(1981), to find the best segment of homology between two sequences.
When using Bestfit or any other sequence alignment program to
determine whether a particular sequence is, for instance, 95%
identical to a reference sequence according to the present
invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5%
of the total number of nucleotides in the reference sequence are
allowed.
[0050] The present application is directed to nucleic acid
molecules at least 70%, 80%, 90%, 95% or 99% identical to the
nucleic acid sequences shown herein in the Sequence Listing which
encode a polypeptide having BAEBL polypeptide activity. By "a
polypeptide having BAEBL activity" is intended polypeptides
exhibiting BAEBL activity in a particular biological assay. For
example, BAEBL protein activity can be measured for changes in
character by an appropriate erythrocyte binding assay.
[0051] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
70%, 80%, 90%, 95%, or 99% identical to a nucleic acid sequence
shown herein in the Sequence Listing will encode a polypeptide
"having BAEBL polypeptide activity." In fact, since degenerate
variants of these nucleotide sequences all encode the same
polypeptide, this will be clear to the skilled artisan even without
performing the above described comparison assay. It will be further
recognized in the art that, for such nucleic acid molecules that
are not degenerate variants, a reasonable number will also encode a
polypeptide having BAEBL polypeptide activity. This is because the
skilled artisan is fully aware of amino acid substitutions that are
either less likely or not likely to significantly effect protein
function (e.g., replacing one aliphatic amino acid with a second
aliphatic amino acid).
[0052] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in (Bowie, J. U. et al.
1990 Science 247: 1306-1310), wherein the authors indicate that
proteins are surprisingly tolerant of amino acid substitutions.
[0053] Polypeptides and Fragments
[0054] The invention further provides a BAEBL polypeptide having
the amino acid sequence encoded by an open reading frame (ORF) of a
wild-type BAEBL gene, or a peptide or polypeptide comprising a
portion thereof (e.g., region II).
[0055] It will be recognized in the art that some amino acid
sequences of the BAEBL polypeptides can be varied without
significant effect of the structure or function of the protein. If
such differences in sequence are contemplated, it should be
remembered that there will be critical areas on the protein which
determine activity.
[0056] Thus, the invention further includes variations of the BAEBL
polypeptide which show substantial BAEBL polypeptide activity or
which include regions of BAEBL protein such as the protein portions
discussed below. Such mutants include deletions, insertions,
inversions, repeats, and type substitutions. As indicated, guidance
concerning which amino acid changes are likely to be phenotypically
silent can be found in (Bowie, J. U. et al. 1990 Science 247:
1306-1310).
[0057] Thus, the fragment, derivative or analog of the polypeptide
of the invention may be (i) one in which one or more of the amino
acid residues are substituted with a conserved or non-conserved
amino acid residue (preferably a conserved amino acid residue) and
such substituted amino acid residue may or may not be one encoded
by the genetic code, or (ii) one in which one or more of the amino
acid residues includes a substituent group, or (iii) one in which
additional amino acids are fused to the mature polypeptide, such as
a fusion peptide or leader or secretory sequence or a sequence
which is employed for purification of the mature polypeptide or a
proprotein sequence. Such fragments, derivatives and analogs are
deemed to be within the scope of those skilled in the art from the
teachings herein.
[0058] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table A).
1TABLE A Conservative Amino Acid Substitutions Aromatic
Phenylalanine Tryptophan Tyrosine Ionizable: Acidic Aspartic Acid
Glutamic Acid Ionizable: Basic Arginine Histidine Lysine
Nonionizable Polar Asparagine Glutamine Serine Threonine Nonpolar
Alanine (Hydrophobic) Glycine Isoleucine Leucine Proline Valine
Sulfur Containing Cysteine Methionine
[0059] Amino acids in the BAEBL polypeptides of the present
invention that are essential for function can be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, 1989 Science
244: 1081-1085). The latter procedure introduces single alanine
mutations at every residue in the molecule. The resulting mutant
molecules are then tested for biological activity such as changes
in erythrocyte binding character.
[0060] The polypeptides of the present invention are conveniently
provided in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a
polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present
invention.
[0061] Also intended as an "isolated polypeptide" are polypeptides
that have been purified, partially or substantially, from a
recombinant host cell or a native source. For example, a
recombinantly produced version of the BAEBL polypeptide can be
substantially purified by the one-step method described in Smith
and Johnson, 1988 Gene 67: 31-40.
[0062] The polypeptides of the present invention include a
polypeptide comprising a polypeptide shown herein in the Sequence
Listing; as well as polypeptides which are at least 70% identical,
and more preferably at least 80%, 90%, 95% or 99% identical to
those described above and also include portions of such
polypeptides.
[0063] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of an
BAEBL polypeptide is intended that the amino acid sequence of the
polypeptide is identical to the reference sequence except that the
polypeptide sequence may include up to five amino acid alterations
per each 100 amino acids of the reference amino acid of the BAEBL
polypeptide. In other words, to obtain a polypeptide having an
amino acid sequence at least 95% identical to a reference amino
acid sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0064] As a practical matter, whether any particular polypeptide is
at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the
amino acid sequence shown herein in the Sequence Listing can be
determined conventionally using known computer programs such the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). When using Bestfit or any
other sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0065] Pharmaceutical Formulations and Modes of Administration
[0066] The compounds of this invention can be employed in admixture
with conventional excipients, i.e., pharmaceutically acceptable
organic or inorganic carrier substances suitable for parenteral,
enteral (e.g., oral) or topical application which do not
deleteriously react with the active compounds. Suitable
pharmaceutically acceptable carriers include but are not limited to
water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl
alcohols, polyethylene glycols, gelatine, carbohydrates such as
lactose, amylose or starch, magnesium stearate, talc, silicic acid,
viscous paraffin, perfume oil, fatty acid monoglycerides and
diglycerides, pentaerythritol fatty acid esters, hydroxy
methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical
preparations can be sterilized and if desired mixed with auxiliary
agents, e.g., lubricants, preservatives, stabilizers, wetting
agents, emulsifiers, salts for influencing osmotic pressure,
buffers, coloring, flavoring and/or aromatic substances and the
like which do not deleteriously react with the active compounds.
They can also be combined where desired with other active agents,
e.g., vitamins.
[0067] For parenteral application, which includes intramuscular,
intradermal, subcutaneous, intranasal, intracapsular, intraspinal,
intrasternal, and intravenous injection, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. Formulations for injection may be
presented in unit dosage form, e.g., in ampoules or in multi-dose
containers, with an added preservative. The compositions may take
such forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0068] For enteral application, particularly suitable are tablets,
dragees, liquids, drops, suppositories, or capsules. The
pharmaceutical compositions may be prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate. A syrup, elixir, or the like can be used
wherein a sweetened vehicle is employed.
[0069] Sustained or directed release compositions can be
formulated, e.g., liposomes or those wherein the active compound is
protected with differentially degradable coatings, e.g., by
microencapsulation, multiple coatings, etc. It is also possible to
freeze dry the new compounds and use the lyophilizates obtained,
for example, for the preparation of products for injection.
[0070] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0071] For topical application, there are employed as non-sprayable
forms, viscous to semi-solid or solid forms comprising a carrier
compatible with topical application and having a dynamic viscosity
preferably greater than water. Suitable formulations include but
are not limited to solutions, suspensions, emulsions, creams,
ointments, powders, liniments, salves, aerosols, etc., which are,
if desired, sterilized or mixed with auxiliary agents, e.g.,
preservatives, stabilizers, wetting agents, buffers or salts for
influencing osmotic pressure, etc. For topical application, also
suitable are sprayable aerosol preparations wherein the active
ingredient, preferably in combination with a solid or liquid inert
carrier material, is packaged in a squeeze bottle or in admixture
with a pressurized volatile, normally gaseous propellant, e.g., a
freon.
[0072] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0073] Reduced Binding to Gerbich Erythrocytes.
[0074] A new gene (baebl) of the DBL-EBP family of Plasmodium
receptor proteins with erythrocyte specificity different from that
of EBA-175 has been studied in P. falciparum. The exon/intron
structure of BAEBL is similar to that of other DBL-EBL (Adams, J.
H. et al. 1992 PNAS USA 89: 7085-7089). The difference between two
DBL-EBP from P. falciparum and P. vivax/P. knowlesi is the
duplication of region II (Adams, J. H. et al. 1992 PNAS USA 89:
7085-7089). Despite the similarity of BAEBL and EBA-175 in their
requirement for sialic acid on erythrocyte proteins, the
specificity of BAEBL for receptors on the erythrocyte surface
differs from that of EBA-175. The differences are two-fold. First,
BAEBL, but not EBA-175, binds to En(a-) erythrocytes that lack
glycophorin A. Second, Gerbich erythrocytes (that have an altered
glycophorin C and absent glycophorin D) bind BAEBL much more weakly
than normal erythrocytes, but bind EBA-175 normally. Thus, the
specificity of these two parasite receptor molecules differs,
suggesting alternative pathways for invasion. These data indicate
at least two different sialic acid-dependent pathways for
invasion.
[0075] In preliminary experiments, BAEBL was not detected in the
parasite supernatant absorbed with glycophorin C/D null
erythrocytes of the Leach phenotype.
[0076] BAEBL, however, was never detected in the eluate of these
erythrocytes. In addition, 2 and 8 .mu.l of Leach erythrocytes, but
not normal erythrocytes, removed BAEBL from parasite supernatant.
The failure to elute BAEBL and its reduction with small numbers of
Leach erythrocytes suggest proteolysis.
[0077] The Gerbich phenotype is found at high allele frequencies
(50%) in some regions of Papua New Guinea (Booth, P. B. et al. 1982
Hum Hered 32: 385-403). The mutation consists of a deletion of exon
3 in glycophorin C that leads to truncated glycophorin C and absent
glycophorin D (Serjeantson, S. W. et al. 1994 Immunol Cell Biol 72:
23-27). Of interest to our study is the fact that these areas
co-localize to hyperendemic areas of malaria. Previously,
Serjeantson (Serjeantson, S. W. 1989 Papua New Guinea Med J 32:
5-9) found reduced frequency of heavy infections with P. falciparum
and P. vivax with the Gerbich phenotype. The common mutation in
erythrocyte band 3, resulting in ovalocytosis, was not described at
the time of the study by Serjeantson (Serjeantson, S. W. 1989 Papua
New Guinea Med J 32: 5-9) and may have influenced the results.
Pasvol et al. (Pasvol, G. et al. Lancet Apr. 21, 1984 907-908)
found reduced invasion of Gerbich erythrocytes, but these were also
ovalocytic (Aanstee, D. J. et al. 1984 Biochem J 218: 615-619). It
is possible that hereditary ovalocytosis caused by band 3 mutations
was influencing the invasion and frequency of infection. It is
critical to restudy these groups now that the two mutations can be
separated by molecular techniques. We were unable to find any
reduction in invasion of Gerbich erythrocytes by the two P.
falciparum clones, Dd2 and Dd2/Nm, but other parasite clones could
be affected. It is known that Dd2 switched to the sialic
acid-independent pathway (Dd2/Nm) after modification of the eba-175
gene (Dolan, S. A. et al. 1990 J Clin Invest 86: 618-624),
suggesting that Dd2 lacking EBA-175 could not invade using BAEBL.
Alternatively, the effect on invasion may be too subtle to be
detected by the invasion assay as performed.
[0078] The tantalizing possibility remains that the high frequency
of the Gerbich phenotype is selected for as a result of reduced
invasion by P. falciparum mediated by the BAEBL receptor. The
Gerbich mutation, like Duffy negativity in West Africa, appears to
be going to fixation in these communities. The difference between
the Gerbich mutation and Duffy negativity is that the Gerbich
negative erythrocytes are still infected by P. falciparum while
Duffy negative erythrocytes are refractory to P. vivax. Still, the
parasite receptors for these two blood groups are envisioned as
being immunogens to prevent malaria because mutation in the host
molecules leads to no infection (P. vivax and Duffy negativity) or
reduced infection (P. falciparum and Gerbich mutation).
[0079] Structure of the baebl Gene.
[0080] BAEBL is predicted to have two cysteine-rich domains
(regions II and VI), a transmembrane region, and a cytoplasmic
region. This structure is characteristic of all DBL-EBP genes
(Adams, J. H. et al. 1992 PNAS USA 89: 7085-7089). The sequence was
obtained from the Sanger Centre chromosome 13 genomic sequence of
P. falciparum clone 3D7
(http://www.sanger.ac.uk/Projects/P.sub.--falciparum/). The
sequence of the gene was also determined from genomic and cDNA
sequences of Dd2/Nm. The exon/intron structure for Dd2/Nm was
identical to that of EBA-175 in that it had four exons: one for the
extracellular domain, one for the transmembrane domain, and two
encoding the cytoplasmic region (FIG. 1). The extracellular exon 1
of Dd2/Nm was identical to the genomic sequence of 3D7 from the
Sanger Centre except for three changes in region II (I185V, N239S,
and K261T; Dd2/Nm amino acid number from GenBank: AF332918/3D7
Sanger Centre chromosome 13). Using microsatellites and a genetic
cross between Dd2 and HB3 (Su, X. et al. 1999 Science 286:
1351-1353), baebl was localized to the end of chromosome 13 close
to marker C13M51.
[0081] Localization and Expression of BAEBL.
[0082] Antibodies to the two cysteine-rich domains (regions II and
VI) for BAEBL of Dd2/Nm were used to determine localization and
expression of the protein. Antibodies to regions II and VI localize
to the same organelle as EBA-175 (FIG. 2A, B), which was previously
shown to localize to the micronemes (Sim, B. K. L. et al. 1992 Mol
Biochem Parasitol 51: 157-160). Furthermore, immunolocalization of
RAP-1, a protein found in rhoptries, another apical organelle of
merozoites, shows that BAEBL is adjacent to but not overlapping
RAP-1 (FIG. 2C, D). This distribution is consistent with the
localization of BAEBL within micronemes, a distribution identical
to EBA-175 and the P. knowlesi Duffy binding protein (Adams, J. H.
et al. 1990 Cell 63: 142-153; Sim, B. K. L. et al. 1992 Mol Biochem
Parasitol 51: 157-160). The fact that antisera against two
different regions of BAEBL showed identical localization within the
parasite indicate that the antisera are not cross-reacting with
another protein.
[0083] To study the molecular characteristics of BAEBL, we used
methods developed for the production of soluble, metabolically
labeled erythrocyte binding proteins (see Examples). Antisera to
regions II and VI immunoprecipitated a protein of approximately 148
kDa; antibodies to region II also immunoprecipitated two lower
molecular weight proteins (129 kDa and 117 kDa). The proof that the
two 135-kDa proteins were identical derived from studies of
immunoabsorption with one sera followed by immunoprecipitation with
the second sera (FIG. 3). The same 135-kDa protein was removed by
both sera, indicating that the antisera to the two regions of BAEBL
were not cross-reacting with another protein. The two lower
molecular weight proteins identified by anti-region II, but not by
anti-region VI, resulted from immunoprecipitation of proteolytic
products of BAEBL that contained region II but not region VI.
[0084] Erythrocyte Binding Specificity.
[0085] We have developed a new assay for measuring binding of BAEBL
to erythrocytes. Previously, EBA-175 was identified in parasite
proteins bound and eluted from human erythrocytes. Its
identification was based on the fact that it was the most abundant
and the highest molecular weight protein eluted from these
erythrocytes. Lower molecular weight proteins may be proteolytic
fragments of EBA-175 or products of different genes. To positively
identify BAEBL, we immunoprecipitated BAEBL from proteins eluted
from erythrocytes with anti-region II and anti-region VI.
[0086] We also modified the protocol in that we identified proteins
removed from the supernatant by erythrocyte absorption. We
determined the different quantities of erythrocytes required to
remove BAEBL from the parasite supernatant. It was found that 25
.mu.l of packed erythrocytes slightly reduced the quantity of
immunoprecipitated BAEBL from 50 .mu.l of parasite supernatant. The
protein was largely removed by absorbing twice with 50 .mu.l of
packed erythrocytes for some culture supernatants and by only one
absorbtion with 50 .mu.l for other supernatants. Therefore, on some
samples, we absorbed with 25 .mu.l, 50 .mu.l once, and 50 .mu.l
twice for comparison between normal erythrocytes and mutant
erythrocytes or enzyme-treated erythrocytes. The protein was also
eluted from the first 50 .mu.l of packed erythrocytes used for
absorption. This set the conditions for absorbing and eluting BAEBL
and demonstrated that BAEBL may be a parasite receptor for binding
erythrocytes.
[0087] To determine the specificity of binding, we studied binding
to neuraminidase-and trypsin-treated human erythrocytes and human
erythrocytes with various genetically modified blood groups. Both
enzymes eliminated the binding of BAEBL to human erythrocytes (FIG.
4), indicating that the erythrocyte receptor required sialic acid
attached to a peptide backbone and must therefore be a
sialoglycoprotein. This failure of neuraminidase-and
trypsin-treated erythrocytes to bind was identical to EBA-175 (FIG.
4). To determine whether BAEBL was binding to the carbohydrates on
the erythrocyte receptor, we performed competitive inhibition with
Neu5Ac(.alpha.2-3) lactosialic and Neu5Ac(.alpha.2-6)lactosialic
acid at 1 .mu.M, 10 .mu.M, 100 .mu.M, and 1000 .mu.M. We determined
that neither Neu5Ac(.alpha.2-3) nor Neu5Ac(.alpha.2-6) lactosialic
acid inhibited the binding of BAEBL to human erythrocytes. These
results indicate that BAEBL is binding either to a more complex
polysaccharide or to a combination of sialic acid and a peptide
backbone of an erythrocyte sialoglycoprotein.
[0088] To further define the binding specificity of BAEBL, we
studied En(a-) erythrocytes, which lack glycophorin A. We found
that EBA-175 failed to bind En(a-) erythrocytes as previously
described (Sim, B. K. L. et al. 1994 Science 264: 1941-1944).
BAEBL, however, bound to these erythrocytes in a similar manner as
to normal erythrocytes (FIG. 5). This demonstrated that the binding
specificity of BAEBL differed from that of EBA-175.
S-s-U-erythrocytes that lacked glycophorin B bound both EBA-175 and
BAEBL. Thus, neither glycophorin A nor B is the sole receptor for
BAEBL.
[0089] Abnormal Binding to Glycophorin C/D Mutant Erythrocytes.
[0090] Another characterized sialoglycoprotein on the surface of
human erythrocytes is glycophorin C/D (Reid, M. E. & Spring, F.
A. 1994 Transfusion Med 4: 139-146; Colin, Y. & Le Van Kim, C.
1995 in: Blood Cell Biochemistry, eds. Cartron, J. P. & Rouger,
P. Plenum Press, New York pp. 331-350). Its peptide backbone is
completely unrelated to glycophorins A and B but, like these, it
has a mucin-like region of serines and threonines for O-linked
sugars at the N-terminus of the protein. Both glycophorin C and
glycophorin D are encoded by the same gene with use of alternative
start codons. Glycophorin C, the full-length protein, contains one
N-linked glycan. There are three mutations of the glycophorin C/D
gene that lack high-incidence antigens (Colin, Y. & Le Van Kim,
C. 1995 in: Blood Cell Biochemistry, eds. Cartron, J. P. &
Rouger, P. Plenum Press, New York pp. 331-350). Leach erythrocytes
are null for these proteins; Gerbich and Yus erythrocytes contain
exon 3 and 2 deletions, respectively, that lead to a shortened
glycophorin C and absent glycophorin D. Both Gerbich and Yus cells
have abnormal N-linked glycosylation of the truncated form of
glycophorin C (Reid, M. E. & Spring, F. A. 1994 Transfusion Med
4: 139-146).
[0091] We screened for the binding of BAEBL to erythrocytes of the
Gerbich (-2, -3, -4) and Yus (-2, -3, -4) phenotype that had been
frozen as pellets in liquid nitrogen. BAEBL had reduced binding to
Gerbich and Yus erythrocytes. These differences were consistent for
pellet-frozen erythrocytes from different donors. EBA-175 bound
normally to these erythrocytes.
[0092] Because the quality of the pellet-frozen erythrocytes was
unpredictable, we obtained fresh blood from a person with the
Gerbich mutation. In two separate experiments, we found that it
required twice as many Gerbich cells to remove BAEBL from the
culture supernatant compared with normal erythrocytes (FIG. 6A). In
contrast to BAEBL, EBA-175 bound equally well to Gerbich and normal
erythrocytes (FIG. 6C). This difference between normal and Gerbich
erythrocytes for absorption of BAEBL was similar to the results
obtained with pellet-frozen erythrocytes as described above.
[0093] BAEBL was eluted from normal but not Gerbich erythrocytes,
indicative of its poor binding to Gerbich erythrocytes (FIG. 6B).
BAEBL also did not elute from neuraminidase-treated normal
erythrocytes. These results are indicative of poor binding of BAEBL
to Gerbich erythrocytes. In contrast to BAEBL, EBA-175 was eluted
from both Gerbich and normal erythrocytes (FIG. 6D).
[0094] Invasion of Gerbich Erythrocytes.
[0095] P. falciparum clones Dd2 and Dd2/Nm invaded Gerbich
erythrocytes at the same rate as normal erythrocytes (Table 1). Dd2
but not Dd2/Nm had markedly reduced invasion into
neuraminidase-treated erythrocytes as described previously (Dolan,
S. A. et al. 1990 J Clin Invest 86: 618-624).
2TABLE 1 Invasion Rate of P. falciparum Into Gerbich Erythrocytes
P. falciparum clones Red-cell type Dd2, % Dd2/Nm, % Normal 3.7* 1.6
Gerbich 3.0 1.8 Neuraminidase-treated normal 0 1.8 Rhesus 0 0
*Percentage of ring-infected erythrocytes.
EXAMPLE 1
[0096] Structure of BAEBL.
[0097] The sequence of BAEBL was identified (Adams J. H., et al.
2001 Trends Parasitol 17: 17297-17299) from cDNA (GenBank No.
N97830) deposited by D. Chakrabarti and from the database supplied
by Sanger for chromosome 13 (>MAL13.sub.--001500, Dec. 27,
2000). Based on this sequence, we sequenced the P. falciparum
clone, Dd2/Nm (Dolan, S. A. et al. 1990 J Clin Invest 86: 618-624)
from genomic DNA (GenBank No. AF332918). The exon/intron boundaries
were defined by RT-PCR of the P. falciparum clone Dd2/Nm (GenBank
No. AF332919 ). Primers use for Dd2/Nm sequencing were:
3 (SEQ ID NO: 3) f1, 5'-AGACCAATAAATTATATATAATGAAAGGA-3' and (SEQ
ID NO: 4) 5'-TTTAAACTTTTCCATTGTTTCTAAACG- -3'; (SEQ ID NO: 5) f2,
5'-ATAAATTTAATTCACTTTCC- GAAAATGA-3' and (SEQ ID NO: 6)
5'-AAAACAATCTCTTCTTTTCCATCAAG-3'; (SEQ ID NO: 7) F3,
5'-TTTATAGGTGATGATATGGATTTTGG-3' and (SEQ ID NO: 8)
5'-TCGTAAATGTTCCAGTACAATTCCT-3'; (SEQ ID NO: 9) f4,
5'-CAAATGGAGGTTTTGACGAACTTG-3' and (SEQ ID NO: 10)
5'-TAAGTACTGCTGACATTACTTTCCA-3'; (SEQ ID NO: 11) f5,
5'-GGAGCTTCAATATATGAGGCGCA-3' and (SEQ ID NO: 12)
5'-ATATCTTCATATTCATTTGGACTCTC-3'; (SEQ ID NO: 13) f6,
5'-TGAGTCATTTAAGGTAGAATGTAAGA-3' and (SEQ ID NO: 14)
5'-GGAACTTTCCGAATGTCCATTCGT-3'; (SEQ ID NO: 15) f7,
5'-TAAATGAACAACAAAGTGGGAAGG- A-3' and (SEQ ID NO: 16)
5'-ATTCTCAATTTGCGTTATATA- TTGATG-3'; (SEQ ID NO: 17) f8,
5'-AGTTCCTTCAGAGGATAATACCCA-3' and (SEQ ID NO: 18)
5'-CTTGATTGACCCTCGCTTTTAAAAC-3'; (SEQ ID NO: 19) f9,
5'-ACTAAAAGAGTAAGGGAGGAAATAAT-3' and (SEQ ID NO: 20)
5'-TATAAAATACATTGAATTATTTAAACTATTG-3'.
[0098] PCR from total RNA untreated with reverse transcriptase
never produced PCR-amplified products. Oligonucleotides
5'-ATTCCTTATTTTGCTGCTGG- AGGT-3' (SEQ ID NO: 21) and
5'-AAGTTGCTTCTATATTAGATTCTCCT-3' (SEQ ID NO: 22) were also used to
sequence fragment f9. Only the 3'-region was sequenced for cDNA to
determine the precise location of the intron/exon boundaries.
[0099] Antisera.
[0100] Antisera to BAEBL region II and region VI of Dd2/Nm were
generated by immunization of rats with a DNA vaccine using the
vector VR1050 (kindly supplied by Stephen Hoffman, Naval Medical
Research Center, Silver Spring, Md.) that contains the T cell
epitopes P2P30 from tetanus toxoid. Region II and region VI gene
fragments of BAEBL were amplified from P. falciparum clone Dd2/Nm
and cloned into VR1050 vector, previously described as
VR1012tPAp2p30 by Becker et al. (Becker, S. I. et al. 1998 Infect
Immun 66: 3457-3461) but now renamed VR1050. The inserts for
regions II and VI of Dd2/Nm spanned from amino acids Q141 to I756
and K1046 to S1132, respectively (GenBank No. AF332918). Rats were
immunized intradermally with 500 .mu.g of DNA at 3-week intervals
for a total of four immunizations. Sera were obtained from the rats
a week after the fourth immunization.
[0101] Rabbit anti-region II of EBA-175 (KLS14) was a kind gift of
David Narum and Kim Lee Sim (EntreMed, Rockville, Md.). Mouse
anti-RAP-1 monoclonal antibody 7H8/50 [MRA-79, Malaria Research and
Reference Reagent Resource (MR4) Center] was a kind gift of Allan
Saul (Queensland Institute of Medical Research, Brisbane,
Australia).
[0102] Erythrocytes Used in the Studies.
[0103] Blood was collected in 10% citrate-phosphate-dextrose
(vol/vol) and stored for up to 4 weeks at 4.degree. C. At the time
of study, the erythrocytes were washed three times in incomplete
media (RPMI-1640; Life Technologies, Rockville, Md.) with 25 mM
HEPES and 0.36 mM hypoxanthine (Sigma, St. Louis, Mo.). For
neuraminidase treatment, 5.5 ml of a 5% (vol/vol) suspension of the
washed human erythrocytes in incomplete media were incubated twice
with 3 milliunits of neuraminidase (Vibrio cholerae; CalBiochem, La
Jolla, Calif.) for 2 hr at 37.degree. C. each time. For trypsin
treatment, washed human erythrocytes were incubated with 1 mg/ml of
tosyl-phenylalanine-chloromethyl-ketone-treated trypsin (Sigma) for
2 hr at 37.degree. C. After trypsin treatment, the cells were
washed once in incomplete medium and incubated with 2 mg/ml soybean
trypsin inhibitor (Sigma) for 10 min at room temperature. The cells
were washed twice before use in a study.
[0104] The glycophorin A and glycophorin B null erythrocytes
[En(a-) and S-s-U-, respectively and the glycophorin D
null/glycophorin C modified erythrocytes (Gerbich cells) were
frozen within a few days of receipt and thawed by the Red Cross
method (Mallory, D. ed. 1993 Immunohematology Methods and
Procedures American Red Cross, National Reference Laboratory,
Rockville, Md., pp. 125-1-125-2). Blood from a Gerbich donor was
collected in 10% (vol/vol) anticoagulant
citrate-phosphate-dextrose.
[0105] Other glycophorin C/D mutant cells (Leach, Gerbich, and Yus
cells) had been stored in liquid nitrogen as frozen pellets (Judd,
W. J. 1994 in: Methods in Immunohematology Montgomery Scientific
Publications Durham, N.C., pp. 188-190) and thawed directly into
PBS at 37.degree. C.
[0106] Metabolic Labeling of Parasite Proteins.
[0107] Soluble, metabolically labeled parasite proteins were
obtained from culture supernatant of schizont-infected erythrocytes
that released merozoites in the absence of uninfected erythrocytes.
The parasites were left to lyse and release proteins into the
culture supernatant. The Dd2/Nm clone of P. falciparum was cultured
as previously described (Kaneko, O. et al. 2000 Mol Biochem
Parasitol 110: 135-14) with the following exceptions.
Schizont-infected erythrocytes (5.times.10.sup.7 per ml of culture
medium) were used during the metabolic labeling. The culture
supernatant was ultracentrifuged in a Beckman Optima TLX
Ultracentrifuge (Beckman, Fullerton, Calif.) at 40,000 rpm
(98,600.times.g) for 10 min at 4.degree. C. before storage at
-70.degree. C.
[0108] Immunoprecipitation.
[0109] Proteins in the supernatant and in the diluted eluate were
immunoprecipitated as previously described (Kaneko, O. et al. 2000
Mol Biochem Parasitol 110: 135-146) with the following exceptions.
The supernatant (50 .mu.l) was diluted into 250 .mu.l of NETT (50
mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.5% Triton X-100)
supplemented with 0.5% bovine serum albumin (BSA; ICN, Aurora,
Ohio). To determine whether the proteins immunoprecipitated by
anti-BAEBL region II and anti-BAEBL region VI are identical, we
preabsorbed with one antisera and immunoprecipitated with the
other. Radiolabeled supernatant (50 .mu.l) was preabsorbed with
protein A-Sepharose as previously described (Kaneko, O. et al. 2000
Mol Biochem Parasitol 110: 135-146). The supernatant was incubated
with 10 .mu.l of anti-BAEBL region II or 10 .mu.l of anti-BAEBL
region VI for 2 hr at 4.degree. C. Protein G-Sepharose (40 .mu.l;
50% vol/vol) was added to remove the immune complexes. Supernatant
was split into two equal volumes and immunoprecipitated with 5
.mu.l of anti-BAEBL region II and 5 .mu.l anti-BAEBL region VI as
described above.
[0110] Modified Erythrocyte Binding Assay.
[0111] Erythrocyte binding assays were developed for metabolically
labeled proteins (as described above) that bind erythrocytes. The
original assay required that parasite proteins be bound and eluted
from some erythrocytes and not from others. In the original study
(Camus, D. & Hadley, T. H. 1985 Science 230: 553-556), the
major protein eluted from the erythrocytes was EBA-175. Lower
molecular proteins could be proteolytic fragments of EBA-175 or
other proteins. Furthermore, this assay is insensitive for less
abundant proteins. Therefore, we have developed a new assay that
depends on the identification of BAEBL with two antisera against
different regions of BAEBL and its removal from the culture
supernatant by human erythrocytes. The parasite protein can also be
identified and quantified by elution of bound protein from
erythrocytes followed by immunoprecipitation. It is then possible
to study its specificity for erythrocyte receptors with
erythrocytes lacking various proteins or with enzymatically
modified erythrocytes. First, we determined the quantity of
erythrocytes that would remove the majority of BAEBL and used this
quantity with erythrocytes of various types (enzyme-modified
erythrocytes and erythrocytes genetically deficient in membrane
proteins) to determine the erythrocyte specificity of BAEBL. We
found that one or two absorptions with a volume of packed
erythrocytes equal to the volume of metabolically labeled
supernatant were required to remove BAEBL from the supernatant,
depending on the concentration of BAEBL in the supernatant.
[0112] Elution from erythrocytes of bound parasite proteins was
performed as described previously (Kaneko, O. et al. 2000 Mol
Biochem Parasitol 110: 135-146). Parasite proteins were eluted only
from the erythrocytes of the first adsorption. The parasite
proteins were eluted as previously described. Because of the
adverse effect of high salt on immunoprecipitation, the eluate was
diluted 5 fold (vol/vol) in NETT with 0.5% BSA prior to
immunoprecipitation.
[0113] Competitive Inhibition Assay.
[0114] An inhibition assay was conducted in the presence of
Neu5Ac(.alpha.2-3) lactosialic acid or Neu5Ac(.alpha.2-6)
lactosialic acid (Sigma). Metabolically labeled parasite
supernatant (50 .mu.l) was preincubated with 1, 10, 100, or 1000
.mu.M in 15 .mu.l of the aforementioned carbohydrates for 1 hr at
room temperature. Packed erythrocytes (50 .mu.l) were added to the
mixture. The erythrocyte binding assay was conducted as described
above. Immunolocalization of BAEBL.
[0115] The methods for immunolocalization of BAEBL by confocal
microscopy were performed as previously described (Kaneko, O. et
al. 2000 Mol Biochem Parasitol 110: 135-146) with the following
modifications. The blocking buffer consisted of PBS (pH 7.4)
containing 0.1% Triton X-100 (Bio-Rad, Hercules, Calif.) and 2.5
mg/ml normal goat serum (Jackson ImmunoResearch Laboratories, West
Grove, Pa.). The secondary antisera consisted of Alexa
488-conjugated goat anti-rat IgG and Alexa 594-conjugated goat
anti-rabbit IgG (Molecular Probes, Eugene, Oreg.) diluted 1:500 in
blocking buffer. For antiquenching, we mounted labeled parasites in
Prolong Antifade (Molecular Probes).
[0116] Invasion Assay.
[0117] Pre-washed A.sup.+ human erythrocytes treated with
neuraminidase and A.sup.+ human erythrocytes of the Gerbich type
(-2, -3, -4) were tested for invasion by P. falciparum clones Dd2
and Dd2/Nm (Dolan, S. A., et al. 1990 J Clin Invest 86: 618-624) as
described previously (Kaneko, O. et al. 1999 Exp Parasitol 93:
116-119). Rhesus (Macaca mulatta) erythrocytes that are resistant
to invasion by P. falciparum were used as a control for normal
erythrocytes introduced with the parasitized erythrocytes.
EXAMPLE 2
[0118] Plasmodium falciparum has evolved great flexibility in its
invasion pathways, in part as a result of multiple copies of the
Duffy binding-like (DBL) family of erythrocyte-binding ligands,
three of which have different red blood cell (RBC) receptor
specificities (Sim, B. K. L. et al. 1994 Science 264: 1941-1944;
Mayer D. C. G. et al. 2001 PNAS USA 98: 5222-5227). This is in
contrast to P. vivax, which has a single copy of the DBL family
member that recognizes the Duffy blood group antigen, explaining
the resistance to P. vivax infection by humans who lack the Duffy
blood group (Chitnis C. E. et al. 1994 J Exp Med 180: 497-506). The
multiplicity of P. falciparum invasion pathways explains the lack
of RBC refractory to invasion. We now describe another mechanism
for recognition of different molecules on the RBC by P. falciparum,
namely, amino acid polymorphisms in the DBL gene, BAEBL, that lead
to different RBC specificities (Adams J. H., et al. 2001 Trends
Parasitol 17: 17297-17299).
[0119] Initial characterization of RBC receptors recognized by
BAEBL from three different P. falciparum clones in three different
laboratories suggested that each had a different RBC receptor (Sim,
B. K. L. et al. 1994 Science 264: 1941-1944; Thompson, J. K. et al.
2001 Mol Microbiol 41: 47-58; Narum, D. L. et al. 2002 Mol Biochem
Parasitol 119: 159-168). We sequenced the baebl gene from eight
parasite clones and found polymorphisms restricted to regions I and
II of the molecules. In total, we sequenced region II of BAEBL from
11 clones from Papua New Guinea (PNG) and 13 clones from other
parts of the world where P. falciparum malaria is highly prevalent.
We observed five different sequence variants in region II with
polymorphisms in four amino acid positions (Table 2). Unlike Africa
where P. falciparum clones were introduced from Asia, PNG P.
falciparum populations are isolated. Surprisingly, the same
sequence variants occurred in PNG as in the rest of the world,
suggesting that mutations leading to these polymorphisms occurred
multiple times. Region II of P. falciparum DBL genes are duplicated
forming the F1 and F2 domains (Adams J. H. et al. 1992 PNAS USA 89:
7085-7089). All base substitutions in the erythrocyte binding
domain of BAEBL occurred in the F1 domain, whereas mutations in
EBA-175, another DBL gene of P. falciparum, were scattered
throughout both F1 and F2 domains and appear not to alter the RBC
binding specificity (Liang, H. & Sim, B. K. 1997 Mol Biochem
Parasitol 84: 241-245).
4TABLE 2 Position of Polymorphisms in BAEBL From Different
Malaria-Endemic Regions Region I* Region II* Clones Origin 26 112
185 239 261 285 PNG2 PNG.dagger. I (ATT) L(CTT) V(GTT) S(AGT)
T(ACG) K(AAA) PNG3 PNG I (ATT) F(TTT) I(ATT) N(AAT) R(AGG) E(GAA)
PNG4 PNG I (ATT) L(CTT) V(GTT) S(AGT) K(AAG) K(AAA) E12 PNG I (ATT)
F(TTT) I(ATT) S(AGT) K(AAG) K(AAA) 1917 PNG I (ATT) L(CTT) V(GTT)
S(AGT) K(AAG) K(AAA) PNG13 PNG I (ATT) F(TTT) V(GTT) S(AGT) K(AAG)
K(AAA) PNG5 PNG I (ATT) L(CTT) I(ATT) S(AGT) K(AAG) K(AAA) 1905 PNG
I (ATT) L(CTT) V(GTT) S(AGT) K(AAG) K(AAA) PNG9-3 PNG I (ATT)
L(CTT) V(GTT) S(AGT) K(AAG) K(AAA) PNG9-1 PNG I (ATT) L(CTT) V(GTT)
S(AGT) T(ACG) K(AAA) PNG10-1 PNG I (ATT) L(CTT) I(ATT) S(AGT)
K(AAG) K(AAA) M24 Kenya I (ATT) F(TTT) I(ATT) N(AAT) K(AAG) K(AAA)
3D7 Africa? I (ATT) F(TTT) I(ATT) N(AAT) K(AAG) K(AAA) Sc/d6 Sierra
Leone I (ATT) L(CTT) V(GTT) S(AGT) K(AAG) K(AAA) Fab9 Kwazulu I
(ATT) L(CTT) I(ATT) N(AAT) R(AGG) E(GAA) Dd2 Vietnam I (ATA) L(CTT)
V(GTT) S(AGT) T(ACG) K(AAA) Camp Malaysia I (ATT) L(CTT) I(ATT)
N(AAT) R(AGG) E(GAA) Dd2/Nm Vietnam I (ATT) L(CTT) V(GTT) S(AGT)
T(ACG) K(AAA) T2/c6 Thailand I (ATT) F(TTT) I(ATT) N(AAT) K(AAG)
K(AAA) MT/S-1 Asia I (ATT) F(TTT) I(ATT) S(AGT) K(AAG) K(AAA) HB3
Honduras I (ATT) L(CTT) V(GTT) S(AGT) K(AAG) K(AAA) PC49 S. America
I (ATT) F(TTT) I(ATT) N(AAT) K(AAG) K(AAA) DIV30 Brazil I (ATT)
L(CTT) V(GTT) S(AGT) T(ACG) K(AAA) PC26 S. America I (ATT) L(CTT)
V(GTT) S(AGT) T(ACG) K(AAA) *For Regions I and II, numbers refer to
the amino acids in the sequence of BAEBL from GenBank AF332918.
Mutated bases and amino acids are shown in bold. .dagger.PNG, Papua
New Guinea.
[0120] We investigated the functional significance of polymorphisms
in the erythrocyte-binding domain of BAEBL. We expressed region II
of four polymorphic groups transiently on the surface of COS cells
with the T8 vector (Buffet P. A. et al. 1999 PNAS USA 96:
12743-12748). This was followed by an erythrocyte-binding assay as
described previously (Chitnis C. E. et al. 1994 J Exp Med 180:
497-506). Binding was performed with normal and enzyme-treated
(trypsin and neuraminidase) erythrocytes and Gerbich-negative
erythrocytes (exon 3 deletion of glycophorin C/D) (Sim, B. K. L. et
al. 1994 Science 264: 1941-1944; Mayer D. C. G. et al. 2001 PNAS
USA 98: 5222-5227). Each of the polymorphisms led to a different
binding specificity as demonstrated by different binding patterns
to enzyme-treated and Gerbich-negative RBC (Table 3). Furthermore,
a single base change led to a change in amino acid and RBC
specificity (e.g., VSTK to VSKK). Such polymorphism in sequence and
receptors was described for influenza hemagglutinin, where a single
base mutation changed the amino acid and the specificity of binding
to sialic acid (Rogers, G. N. et al. 1983 Nature 304: 76-78).
5TABLE 3 Binding Patterns of BAEBL Variants to Enzyme-Treated and
Gerbich-Negative RBC Region II Normal RBC Gerbich-negative
variants* Untreated.sup..dagger. Trypsin.sup..dagger-dbl.
Neuraminidase.sup..dagger-dbl. RBC.sup..dagger-dbl. VSTK 65 0 0
0.9% VSKK 58 0 90% 112% ISKK 59 92% 0 96% INRE 67 114% 110% 100%
*Region II of BAEBL expressed in COS cells is from amino acid 143
to 606 (GenBank AF332918) and contains the mutations shown at the
positions delineated in Table 2. .sup..dagger.COS cells with five
or more attached RBC were counted and the total per coverslip
recorded. .sup..dagger-dbl.Data from enzyme-treated and
Gerbich-negative RBC are expressed as the percentage of binding to
normal, untreated RBC.
[0121] The Gerbich-negative phenotype occurs at an allelic
frequency of 50% in some regions of PNG (Booth, P. B. et al. 1982
Human Hered 32: 385-403). It is tantalizing to postulate that the
polymorphisms in BAEBL could be a coevolutionary adaptation to the
disappearance of the RBC receptor in Gerbich-negative individuals.
Why then has the Gerbich-negative phenotype not been described in
all the geographic areas where mutations in region II of BAEBL have
occurred? One possibility is that the Gerbich-negative phenotype
could indeed be more widespread than previously described.
Alternatively, such polymorphisms in BAEBL receptor specificity may
be advantageous to the parasite independently of the Gerbich
negative phenotype.
[0122] While the present invention has been described in some
detail for purposes of clarity and understanding, one skilled in
the art will appreciate that various changes in form and detail can
be made without departing from the true scope of the invention. All
figures, tables, and appendices, as well as patents, applications,
and publications, referred to above, are hereby incorporated by
reference.
Sequence CWU 1
1
22 1 4138 DNA Plasmodium falciparum 1 tttataatat cgttttttta
tgagcattat atcatataaa taattaatta aataaatata 60 tatatatata
taattataat tagaccaata aattatatat aatgaaagga tattttaata 120
tatatttttt aattccttta atttttttat ataatgtaat aagaataaat gaatcaataa
180 taggtagaac actttataat agacaagatg aatcatcaga tatttcaagg
gtaaattcac 240 ccgaattaaa taataatcat aaaactaata tatatgattc
agattacgaa gatgtaaata 300 ataaattaat aaacagtttt gtagaaaata
aaagtgtgaa aaaaaaaagg tctttaagtt 360 ttataaataa taaaacaaaa
tcatatgata taattccacc ttcatattca tataggaatg 420 ataaatttaa
ttcactttcc gaaaatgaag ataattctgg aaatacaaat agtaataatt 480
tcgcaaatac ttctgaaata tctattggaa aggataataa acaatatacg tttatacaga
540 aacgtactca tttgtttgct tgtggaataa aaagaaaatc aataaaatgg
atatgtcgag 600 aaaacagtga gaaaattact gtatgtgttc ctgatagaaa
aatacaacta tgtgttgcaa 660 attttttaaa ctcacgttta gaaacaatgg
aaaagtttaa agaaatattt ttaatttctg 720 ttaatacaga agcaaaatta
ttatataaca aaaatgaagg aaaagatccc tcaatatttt 780 gtaatgaatt
aagaaatagt ttttcagatt ttagaagttc atttataggt gatgatatgg 840
attttggtgg taatacagat agagtcaaag gatatattaa tacgaagttc tccgattatt
900 ataaggaaaa aaatgttgaa aaattaaata atatcaaaaa agaatggtgg
gaaaaaaata 960 aagcaaattt gtggaatcac atgatagtaa atcataaagg
aaacataagt aaagaatgtg 1020 ccataattcc cgcggaagaa cctcaaatta
atctatggat aaaagaatgg aatgaaaact 1080 tcttgatgga aaagaagaga
ttgtttttaa atataaaaga taagtgtgtt gaaaacaaaa 1140 aatatgaagc
atgttttggt ggatgtaggc ttccatgttc ttcatataca tcatttatga 1200
aaaaaagtaa aacacaaatg gaggttttga cgaacttgta taaaaagaaa aattcaggag
1260 tggataaaaa taattttctg aatgatcttt ttaaaaaaaa taataaaaat
gatttagatg 1320 attttttcaa aaatgaaaag gaatatgatg atttatgtga
ttgcagatat actgctacta 1380 ttattaaaag ttttctaaat ggtcctgcta
aaaatgatgt agatattgca tcacaaatta 1440 atgttaatga tcttcgaggg
tttggatgta attataaaag taataatgaa aaaagttgga 1500 attgtactgg
aacatttacg aacaaatttc ctggtacatg tgaacccccc agaagacaaa 1560
ctttatgtct tggacgtaca tatcttttac atcgtggtca tgaggaagat tataaggaac
1620 atttacttgg agcttcaata tatgaggcgc aattattaaa atataaatat
aaggaaaagg 1680 atgaaaatgc attgtgtagt ataatacaaa atagttatgc
agatttggca gatattatca 1740 agggatcgga tataataaaa gattattatg
gtaaaaaaat ggaagaaaat ttaaataaag 1800 taaacaaaga taaaaaacgt
aatgaagaat ctttgaagat ttttcgtgaa aaatggtggg 1860 atgaaaacaa
ggagaatgta tggaaagtaa tgtcagcagt acttaaaaat aaggaaacgt 1920
gtaaagatta tgataagttt caaaagattc ctcaattttt aagatggttt aaggaatggg
1980 gagacgattt ttgtgagaaa agaaaagaga aaatatattc atttgagtca
tttaaggtag 2040 aatgtaagaa aaaagattgt gatgaaaata catgtaaaaa
taaatgtagt gaatataaaa 2100 aatggataga tttgaaaaaa agtgaatatg
agaaacaagt tgataaatac acaaaagata 2160 aaaataaaaa gatgtatgat
aatattgatg aagtaaaaaa taaagaagcc aatgtttact 2220 taaaagaaaa
atccaaagaa tgtaaagatg taaatttcga tgataaaatt tttaatgaga 2280
gtccaaatga atatgaagat atgtgtaaaa aatgtgatga aataaaatat ttaaatgaaa
2340 ttaaatatcc taaaacaaaa cacgatatat atgatataga tacattttca
gatacttttg 2400 gtgatggaac gccaataagt attaatgcaa atataaatga
acaacaaagt gggaaggata 2460 cctcaaatac tggaaatagt gaaacatcag
attcaccggt tagtcatgaa ccagaaagtg 2520 atgctgcaat taatgtagaa
aagttaagtg gtgatgaaag ttcaagtgaa acaagaggaa 2580 tattagatat
taatgatcca agtgttacga acaatgtcaa tgaagttcat gatgcttcaa 2640
atacacaagg tagtgtttca aatacttctg atataacgaa tggacattcg gaaagttccc
2700 tgaatagaac aacgaatgca caagatatta aaataggccg ttcaggaaat
gaacaaagtg 2760 ataatcaaga aaatagttca cattctagtg ataattcagg
ttctttgaca atcggacaag 2820 ttccttcaga ggataatacc caaaatacat
atgattcaca aaaccctcat agagatacac 2880 ctaatgcatt agcatcttta
ccatcagatg ataaaattaa tgaaatagag ggtttcgatt 2940 ctagtagaga
tagtgaaaat ggtaggggtg atacaacatc aaatactcat gatgtacgtc 3000
gtacgaatat agtaagtgag agacgtgtga atagccatga ttttattaga aacggaatgg
3060 cgaataacaa tgcacatcat caatatataa cgcaaattga gaataatgga
atcataagag 3120 gacaagagga aagtgcgggg aatagtgtta attataaaga
taatccaaag aggagtaatt 3180 tttcctccga aaatgatcat aagaaaaata
tacaggaata taattctaga gatactaaaa 3240 gagtaaggga ggaaataatt
aaattatcga agcaaaataa atgcaacaat gaatattcca 3300 tggaatattg
tacctattct gacgaaagga atagttcacc gggtccttgt tctagagaag 3360
aaagaaagaa attatgttgt cagatttcag attattgttt aaaatatttt aacttttatt
3420 caattgaata ttataattgt ataaaatctg aaattaaaag tccagaatat
aaatgtttta 3480 aaagcgaggg tcaatcaagt atgtttcata tataatgaaa
ataactaaat taaaataaat 3540 atctaaattt tccatttaaa tataatttaa
ataatttatt tttttattat aaaatttatt 3600 tgtttttttt tatataatgt
tttatttttt ctttacaggc attccttatt ttgctgctgg 3660 aggtatttta
gttgtaatag tcttactttt gagttcagca tctagaatgg ggaaaaggtt 3720
acacgcataa gattaaataa atatgactat actttttaat ttttatatta tttatattat
3780 tattatgttt taacaagaat ataaattata ttatatattt atatgtatat
atattttttt 3840 atatagtaat gaagaatatg atataggaga atctaatata
gaagcaactt ttgaagaaaa 3900 taattattta aataaactat cgcgcatatg
taagattaat ataaaaagtg aaaatttcat 3960 attaaataaa taaatgaata
tatatatata tatatatata tatatatata tatatacatg 4020 ttatataaat
tttaatgtta tattatttac ttttttctca gttaatcaag aagtacaaga 4080
gacaaacatt tcagattatt ccgagtacaa ttataatgaa aagaatatgt attaattt
4138 2 1210 PRT Plasmodium falciparum 2 Met Lys Gly Tyr Phe Asn Ile
Tyr Phe Leu Ile Pro Leu Ile Phe Leu 1 5 10 15 Tyr Asn Val Ile Arg
Ile Asn Glu Ser Ile Ile Gly Arg Thr Leu Tyr 20 25 30 Asn Arg Gln
Asp Glu Ser Ser Asp Ile Ser Arg Val Asn Ser Pro Glu 35 40 45 Leu
Asn Asn Asn His Lys Thr Asn Ile Tyr Asp Ser Asp Tyr Glu Asp 50 55
60 Val Asn Asn Lys Leu Ile Asn Ser Phe Val Glu Asn Lys Ser Val Lys
65 70 75 80 Lys Lys Arg Ser Leu Ser Phe Ile Asn Asn Lys Thr Lys Ser
Tyr Asp 85 90 95 Ile Ile Pro Pro Ser Tyr Ser Tyr Arg Asn Asp Lys
Phe Asn Ser Leu 100 105 110 Ser Glu Asn Glu Asp Asn Ser Gly Asn Thr
Asn Ser Asn Asn Phe Ala 115 120 125 Asn Thr Ser Glu Ile Ser Ile Gly
Lys Asp Asn Lys Gln Tyr Thr Phe 130 135 140 Ile Gln Lys Arg Thr His
Leu Phe Ala Cys Gly Ile Lys Arg Lys Ser 145 150 155 160 Ile Lys Trp
Ile Cys Arg Glu Asn Ser Glu Lys Ile Thr Val Cys Val 165 170 175 Pro
Asp Arg Lys Ile Gln Leu Cys Val Ala Asn Phe Leu Asn Ser Arg 180 185
190 Leu Glu Thr Met Glu Lys Phe Lys Glu Ile Phe Leu Ile Ser Val Asn
195 200 205 Thr Glu Ala Lys Leu Leu Tyr Asn Lys Asn Glu Gly Lys Asp
Pro Ser 210 215 220 Ile Phe Cys Asn Glu Leu Arg Asn Ser Phe Ser Asp
Phe Arg Ser Ser 225 230 235 240 Phe Ile Gly Asp Asp Met Asp Phe Gly
Gly Asn Thr Asp Arg Val Lys 245 250 255 Gly Tyr Ile Asn Thr Lys Phe
Ser Asp Tyr Tyr Lys Glu Lys Asn Val 260 265 270 Glu Lys Leu Asn Asn
Ile Lys Lys Glu Trp Trp Glu Lys Asn Lys Ala 275 280 285 Asn Leu Trp
Asn His Met Ile Val Asn His Lys Gly Asn Ile Ser Lys 290 295 300 Glu
Cys Ala Ile Ile Pro Ala Glu Glu Pro Gln Ile Asn Leu Trp Ile 305 310
315 320 Lys Glu Trp Asn Glu Asn Phe Leu Met Glu Lys Lys Arg Leu Phe
Leu 325 330 335 Asn Ile Lys Asp Lys Cys Val Glu Asn Lys Lys Tyr Glu
Ala Cys Phe 340 345 350 Gly Gly Cys Arg Leu Pro Cys Ser Ser Tyr Thr
Ser Phe Met Lys Lys 355 360 365 Ser Lys Thr Gln Met Glu Val Leu Thr
Asn Leu Tyr Lys Lys Lys Asn 370 375 380 Ser Gly Val Asp Lys Asn Asn
Phe Leu Asn Asp Leu Phe Lys Lys Asn 385 390 395 400 Asn Lys Asn Asp
Leu Asp Asp Phe Phe Lys Asn Glu Lys Glu Tyr Asp 405 410 415 Asp Leu
Cys Asp Cys Arg Tyr Thr Ala Thr Ile Ile Lys Ser Phe Leu 420 425 430
Asn Gly Pro Ala Lys Asn Asp Val Asp Ile Ala Ser Gln Ile Asn Val 435
440 445 Asn Asp Leu Arg Gly Phe Gly Cys Asn Tyr Lys Ser Asn Asn Glu
Lys 450 455 460 Ser Trp Asn Cys Thr Gly Thr Phe Thr Asn Lys Phe Pro
Gly Thr Cys 465 470 475 480 Glu Pro Pro Arg Arg Gln Thr Leu Cys Leu
Gly Arg Thr Tyr Leu Leu 485 490 495 His Arg Gly His Glu Glu Asp Tyr
Lys Glu His Leu Leu Gly Ala Ser 500 505 510 Ile Tyr Glu Ala Gln Leu
Leu Lys Tyr Lys Tyr Lys Glu Lys Asp Glu 515 520 525 Asn Ala Leu Cys
Ser Ile Ile Gln Asn Ser Tyr Ala Asp Leu Ala Asp 530 535 540 Ile Ile
Lys Gly Ser Asp Ile Ile Lys Asp Tyr Tyr Gly Lys Lys Met 545 550 555
560 Glu Glu Asn Leu Asn Lys Val Asn Lys Asp Lys Lys Arg Asn Glu Glu
565 570 575 Ser Leu Lys Ile Phe Arg Glu Lys Trp Trp Asp Glu Asn Lys
Glu Asn 580 585 590 Val Trp Lys Val Met Ser Ala Val Leu Lys Asn Lys
Glu Thr Cys Lys 595 600 605 Asp Tyr Asp Lys Phe Gln Lys Ile Pro Gln
Phe Leu Arg Trp Phe Lys 610 615 620 Glu Trp Gly Asp Asp Phe Cys Glu
Lys Arg Lys Glu Lys Ile Tyr Ser 625 630 635 640 Phe Glu Ser Phe Lys
Val Glu Cys Lys Lys Lys Asp Cys Asp Glu Asn 645 650 655 Thr Cys Lys
Asn Lys Cys Ser Glu Tyr Lys Lys Trp Ile Asp Leu Lys 660 665 670 Lys
Ser Glu Tyr Glu Lys Gln Val Asp Lys Tyr Thr Lys Asp Lys Asn 675 680
685 Lys Lys Met Tyr Asp Asn Ile Asp Glu Val Lys Asn Lys Glu Ala Asn
690 695 700 Val Tyr Leu Lys Glu Lys Ser Lys Glu Cys Lys Asp Val Asn
Phe Asp 705 710 715 720 Asp Lys Ile Phe Asn Glu Ser Pro Asn Glu Tyr
Glu Asp Met Cys Lys 725 730 735 Lys Cys Asp Glu Ile Lys Tyr Leu Asn
Glu Ile Lys Tyr Pro Lys Thr 740 745 750 Lys His Asp Ile Tyr Asp Ile
Asp Thr Phe Ser Asp Thr Phe Gly Asp 755 760 765 Gly Thr Pro Ile Ser
Ile Asn Ala Asn Ile Asn Glu Gln Gln Ser Gly 770 775 780 Lys Asp Thr
Ser Asn Thr Gly Asn Ser Glu Thr Ser Asp Ser Pro Val 785 790 795 800
Ser His Glu Pro Glu Ser Asp Ala Ala Ile Asn Val Glu Lys Leu Ser 805
810 815 Gly Asp Glu Ser Ser Ser Glu Thr Arg Gly Ile Leu Asp Ile Asn
Asp 820 825 830 Pro Ser Val Thr Asn Asn Val Asn Glu Val His Asp Ala
Ser Asn Thr 835 840 845 Gln Gly Ser Val Ser Asn Thr Ser Asp Ile Thr
Asn Gly His Ser Glu 850 855 860 Ser Ser Leu Asn Arg Thr Thr Asn Ala
Gln Asp Ile Lys Ile Gly Arg 865 870 875 880 Ser Gly Asn Glu Gln Ser
Asp Asn Gln Glu Asn Ser Ser His Ser Ser 885 890 895 Asp Asn Ser Gly
Ser Leu Thr Ile Gly Gln Val Pro Ser Glu Asp Asn 900 905 910 Thr Gln
Asn Thr Tyr Asp Ser Gln Asn Pro His Arg Asp Thr Pro Asn 915 920 925
Ala Leu Ala Ser Leu Pro Ser Asp Asp Lys Ile Asn Glu Ile Glu Gly 930
935 940 Phe Asp Ser Ser Arg Asp Ser Glu Asn Gly Arg Gly Asp Thr Thr
Ser 945 950 955 960 Asn Thr His Asp Val Arg Arg Thr Asn Ile Val Ser
Glu Arg Arg Val 965 970 975 Asn Ser His Asp Phe Ile Arg Asn Gly Met
Ala Asn Asn Asn Ala His 980 985 990 His Gln Tyr Ile Thr Gln Ile Glu
Asn Asn Gly Ile Ile Arg Gly Gln 995 1000 1005 Glu Glu Ser Ala Gly
Asn Ser Val Asn Tyr Lys Asp Asn Pro Lys Arg 1010 1015 1020 Ser Asn
Phe Ser Ser Glu Asn Asp His Lys Lys Asn Ile Gln Glu Tyr 1025 1030
1035 1040 Asn Ser Arg Asp Thr Lys Arg Val Arg Glu Glu Ile Ile Lys
Leu Ser 1045 1050 1055 Lys Gln Asn Lys Cys Asn Asn Glu Tyr Ser Met
Glu Tyr Cys Thr Tyr 1060 1065 1070 Ser Asp Glu Arg Asn Ser Ser Pro
Gly Pro Cys Ser Arg Glu Glu Arg 1075 1080 1085 Lys Lys Leu Cys Cys
Gln Ile Ser Asp Tyr Cys Leu Lys Tyr Phe Asn 1090 1095 1100 Phe Tyr
Ser Ile Glu Tyr Tyr Asn Cys Ile Lys Ser Glu Ile Lys Ser 1105 1110
1115 1120 Pro Glu Tyr Lys Cys Phe Lys Ser Glu Gly Gln Ser Ser Ile
Pro Tyr 1125 1130 1135 Phe Ala Ala Gly Gly Ile Leu Val Val Ile Val
Leu Leu Leu Ser Ser 1140 1145 1150 Ala Ser Arg Met Gly Lys Ser Asn
Glu Glu Tyr Asp Ile Gly Glu Ser 1155 1160 1165 Asn Ile Glu Ala Thr
Phe Glu Glu Asn Asn Tyr Leu Asn Lys Leu Ser 1170 1175 1180 Arg Ile
Phe Asn Gln Glu Val Gln Glu Thr Asn Ile Ser Asp Tyr Ser 1185 1190
1195 1200 Glu Tyr Asn Tyr Asn Glu Lys Asn Met Tyr 1205 1210 3 29
DNA Artificial Sequence primer 3 agaccaataa attatatata atgaaagga 29
4 27 DNA Artificial Sequence primer 4 tttaaacttt tccattgttt ctaaacg
27 5 28 DNA Artificial Sequence primer 5 ataaatttaa ttcactttcc
gaaaatga 28 6 26 DNA Artificial Sequence primer 6 aaaacaatct
cttcttttcc atcaag 26 7 26 DNA Artificial Sequence primer 7
tttataggtg atgatatgga ttttgg 26 8 25 DNA Artificial Sequence primer
8 tcgtaaatgt tccagtacaa ttcct 25 9 24 DNA Artificial Sequence
primer 9 caaatggagg ttttgacgaa cttg 24 10 25 DNA Artificial
Sequence primer 10 taagtactgc tgacattact ttcca 25 11 23 DNA
Artificial Sequence primer 11 ggagcttcaa tatatgaggc gca 23 12 26
DNA Artificial Sequence primer 12 atatcttcat attcatttgg actctc 26
13 26 DNA Artificial Sequence primer 13 tgagtcattt aaggtagaat
gtaaga 26 14 24 DNA Artificial Sequence primer 14 ggaactttcc
gaatgtccat tcgt 24 15 25 DNA Artificial Sequence primer 15
taaatgaaca acaaagtggg aagga 25 16 27 DNA Artificial Sequence primer
16 attctcaatt tgcgttatat attgatg 27 17 24 DNA Artificial Sequence
primer 17 agttccttca gaggataata ccca 24 18 25 DNA Artificial
Sequence primer 18 cttgattgac cctcgctttt aaaac 25 19 26 DNA
Artificial Sequence primer 19 actaaaagag taagggagga aataat 26 20 31
DNA Artificial Sequence primer 20 tataaaatac attgaattat ttaaactatt
g 31 21 24 DNA Artificial Sequence oligonucleotide 21 attccttatt
ttgctgctgg aggt 24 22 26 DNA Artificial Sequence oligonucleotide 22
aagttgcttc tatattagat tctcct 26
* * * * *
References